Compound for restoring radiation injury and process for preparation thereof

ABSTRACT

COMPOUNDS FOR RESTORING RADIATION INJURY WHICH ARE THE REACTION PRODUCT OF A BISIC POLYAMINE OR BASIC POLYAMINO ACID AND DEXOYRIBONUCLEI ACID, AND A PROCESS FOR PREPARATION THEREOF.

United States Patent Olfiee 3,803,] 16 Patented Apr. 9, 1974 3,803,116COMPOUND FOR RESTORING RADIATION INJURY AND PROCESS FOR PREPARA- TIONTHEREOF Toyozo Sekiguchi and Tomio Fujii, Tokyo-t0, and Hldeo Kobayashi,Chiba, Japan, assignors to Kakenyaku Kako Kabushiki Kaisha, Tokyo-to,Japan No Drawing. Filed May 27, 1970, Ser. No. 41,045 Int. Cl. C07d51/50 US. Cl. 260-1125 4 Claims ABSTRACT OF THE DISCLOSURE Compounds forrestoring radiation injury which are the reaction product of a basicpolyamine or basic polyamino acid and deoxyribonuclei acid, and aprocess for preparation thereof.

DETAILED EXPLANATION OF THE INVENTION This invention relates tocompounds effective for restoring and protection against radiationinjury composed of the reaction product of a basic polyamine anddeoxyribonucleic acid -(-DNA) and to a process for preparing the same.

It is generally known that the biosynthesis DNA in the cells of humanbeings and mammals is severely damaged by ionizing radiation. Also it isrecognized that such biosynthesis of DNA is indispensable for celldivision, and thus the inhibition of DNA biosynthesis makes impossiblecell division and finally leads to the death of the cells.

Recently diagnosis and treatment in the early stage of cancer have drawnstrong attention in the medical field and have widened the use ofvarious treatments such as those with ionizing radiation,pharmacological preparations or surgical operation.

However, radiation treatment or pharmacological treatment are generallyaccompanied by several kinds of side effects, particularly inhematologic organs where the cell division is most active. Thus, it isfrequently impossible to continue the cancer treatment due to seriousside effects such as leucopenia, the abnormal decrease of blood whitecells.

Consequently a suitable substance capable of restoring and protectingagainst radiation injury is strongly desired, and such substance isexpected to be able to restore the damage caused by the side elfects ofcancer treatment.

However, there are some protective drugs such as sulphydryl containingcompounds which are reactive with radiation induced free radicals whichhave been formed in the human body by ionizing radiation and are onlyeffective when administered to the human beings before X-rayirradiation. But there are very few drugs which can restore theradiation injury when administered after the irradiation. Such drugs arecalled as restorator and differentiate with the former drugs, theprotecters.

Today, it is a restorer that is strongly desired to restore the severeside efiects accompanying cancer treatment by ionizing radiation such asX-rays or cobalt gamma rays.

At present, however, substances capable of restoring the inhibition ofthe DNA biosynthesis are scarcely known, and only confirmed is the factthat high molecular weight DNA isolated from the same species iseffective in restoring the radiation injury in irradiated cells ofanimals.

However, it is quite diflicult to use a DNA preparation which isisolated from human beings to restore human radiation injury because itis quite difiicult to obtain such human tissue materials as DNA source.In the practical use of a DNA preparation as a pharmacological drug itmust be considered that the DNA must be isolated from a species otherthan human beings.

Furthermore, there may be some possibility of hereditary danger if therebe used very high molecular weight foreign DNA derived from anotherspecies as a pharmacological drug, thus making it necessary to use lowermolecular weight DNA ranging from 200,000 to 500,000 to avoid suchhereditary danger of genetic mutation because it is well known that suchlower molecular weight DNA cannot induce the genetic transformation evenin the bacterial system.

It is well known that in the higher animals DNA is not present in thefree state but combined with histone, a basic protein.

Also there is a possibility that the inhibition of DNA biosynthesis orstrand scission of DNA double helices resulting from radiation may becaused secondarily by damage to basic protein combined with DNA.

Therefore, it is not only the inhibition of DNA biosynthesis orrestoring the damage to the DNA molecule which must be considered butalso the restoring of the damage to histone, a basic protein combined toDNA molecule and modified the biological function of DNA, are quitenecessary to obtain the more effective restoration of the radiationinjury of the irradiated mammals.

It is quite impossible to use foreign histone from foreign sources torestore the radiation injury to the histone moiety, however, because itcannot be avoided that severe allergic reaction would be induced whenforeign protein was administered (antigen-antibody reaction).

On the other hand it is confirmed that lower molecular weight polyaminoacids or polyamines having molecular weights of several hundreds cannotinduce antigen-antibody reactions.

Based on the facts mentioned above, the present in'ven tors conceivedthat the administration of artificial compounds of DNA combined withantigenetically safe basic polyamines or basic polyamino acidsresembling the na tural complex compound of DNA and basic protein,histone, would be more effective than the simple administration of DNAalone against radiation injury and tried to produce various compoundsfrom lower molecular weight DNA and several kinds of basic polyamine orbasic polyamino acid,

Thus it is an object of this invention to provide a process of producingcompounds from DNA and basic polyamines or basic polyamino acidscomprising the steps of dissolving DNA having molecular weight rangingfrom 200,000 to 500,000 isolated from sperm of fish such as salmon ortrout or from internal organs such as thymus, liver or kidney of mammalssuch as calf, or young pig and basic polyamines or basic polyaminoacids, the weight of polyamine or polyamino acid being V to ,6 of theweight of, separately in dilute sodium chloride solution (0.0015 N) orin a mixed solution of sodium chloride and sodium citrate (0.0015 N and0.00015 M). Then the pH of each solution is adjusted exactly to pH 7.0'by adding 0.1 N sodium hydroxide solution or 0.1 N hydrochloric acid,then the solutions are mixed and reacted, allowing the mixture to standat 37 C. for ca. 6 to 36 hours. Then there was added 99% ethanol to thereaction mixture in a volume about 5 times that of the mixture, and themixture was allowed to stand for 30 minutes, the resulting whiteprecipitate by eentrifugation of at 5,000 r.p.m. for 10 minutes. Twicethe volume of 99% ethanol was added to the collected precipitate underagitation, the mixture was again subjected to centrifugation at 5,000rpm. for 10 minutes and the collected precipitate was recrystallizedfrom ethanol or ethyl ether to obtain the DNAzbasic polyamine or basicpolyamino acid compound.

Physical and chemical properties of the compound constituted from DNAand basic polyamine or polyamino acid according to the process of thisinvention are as follows:

(1) Molecular weight, 200,000 to 500,000

(2) Sedimentation constant, 12s or higher (3) Specific gravity,1.6501.700

(4) Viscosity, n =3 X 10 -6 X 10- (5 Specific optical rotation, tan a=5l0- 10" (6) Absorption spectra, B =2600 A.-2657 A.

(7) Appearance, colorless crystalline powder (8) Solubility, soluble inwater, dilute saline (0.015 N insoluble in ethanol, acetone and ethylether.

The restorative efieet on radiation injury by the product according tothis invention was tested by two methods.

First, there was employed the colony forming method using tissuecultured cells which consists of cultivating the cells, human originChangs liver cells, with Eagles MEM (minimum essential media)supplemented with 10% of calf serum in a glass petri dish for two weeksto form visible colonies thereby evaluating the restoration of theirradiated cells from radiation injury. Since dead cells are incapableof forming a visible colony, restorative effects of the compoundsobtained according to this invention were evaluated by observing thesurvival ratio of the irradiated cells by counting the number of visiblecolonies. Changs liver cells were subjected to the radiation of 200,400, 600 and 800 R. with X-ray of 300 kvp., and the number of cellssurviving after two weeks was determined by the colony count.

In comparison with the control group to which DNA was not added, thegroups to which DNA alone was added showed a slight increase, ca. 1.5times, of the number of colonies at each radiation dose.

On the other hand the complex compound of DNA and spermidine which isone of the basic polyamines which may be used according to the inventionshowed. marked effectiveness when it was added to the culture mediaimmediately after the X-ray irradiation of the cells.

Namely, the number of colonies was increased to about four times that inthe control group.

Secondly, the restorative effect of the compounds of this invention wastested by animal experiment using mice the whole bodies of which wereirradiated with X-rays.

The mice used were strain ddn, female, and the body weight ranged fromto g. Radiation factors were 550 R. for the whole body irradiation byX-rays of 300 kvp.

In the control group the mice were intraperitoneally injectedimmediately after the X-ray irradiation with dilute saline (0.015 M NaCland 0.0015 M sodium citrate solution) which was the solvent for DNA orthe DNA basic polyamine complex compound. The injections were repeatedthree times a week.

In the second group were injected intraperitoneally immediately afterirradiation with herring sperm DNA having a molecular weight of 200,000at a dose of 25 ug./ g. body weight of the mouse. The injections wererepeated three times a week for four weeks.

In the third group the mice were injected with the complex compound ofherring sperm DNA having a molecular weight of 200,000 and spermidine, abasic polyamine. In the complex compound the weight ratio of DNA tospermidine was 50:1.

The dose of complex intraperitoneally injected immediately after theirradiation was 25 ,ug. of DNA equivalent for each gram of 'body weightand the injections were repeated three times a week for four weeks.

In the fourth group the mice were injected with the complex compound ofthe same DNA and spermidine, but the weight ratio of DNA to spermidinewas 25:1. The injection was carried out for the second group.

In the fifth group the mice were injected with the complex compound ofthe same DNA and spermidine, but

4- the weight ratio of DNA to spermidine was 10:1. The injection wascarried out as for the second group.

The results of the tests are shown by the following Table 1.

Injection with DNA alone was almost inefiective in the survival ratio asshown by the mice experiments in which the mice were subjected overtheir entire bodies to an exposure dose of X-rays greater than LD But,it was found that DNA and spermidine compounds in various weight ratiosshowed upon injection an increase of more than twice in the survivalratio and an extended average life span of about a week, this beingshown in the animal experiments, in comparison with the control group.

EXAMPLE l 0.5 g. of spermine (C H N molecular weight 202.27) and 10 g.of DNA having molecular weight of ca. 500,000 extracted from salmonsperm were separately dissolved into the mixed solution of 0.01 M sodiumchloride and 0.014 M of the sodium citrate and each of the resultingsolutions was adjusted to pH 7.0 by adding 0.1 N sodium hydroxide or 0.1N hydrochloric acid. Then these solutions were well mixed to effectreaction and allowed to stand at 37 C. for 18 hours. Then, 5 volumes of99% ethyl alcohol was added to the mixture and allowed to stand for 30minutes. The resulting white precipitate was collected by centrifugationat 5,000 r.p.m. for 10 minutes, then added with twice the volume of 99%ethyl alcohol under agitation and again subjected to centrifugation at5,000 r.p.m. for 10 minutes. The precipitate thus collected isrecrystallized from ethyl alcohol to obtain 10.4 g. of colorlesscrystalline powder.

EXAMPLE 2 0.2 g. of spermidine trihydrochloride (C H N molecuar weight145.25) and 10 g. of DNA having a molecular weight of ca. 500,000extracted from herring sperm were separately dissolved into a solutionof a mixture of 0.015 M sodium chloride and 0.0015 M sodium citrate andthe resulting solutions were adjusted to pH 7.0 by adding 0.1 N sodiumhydroxide solution or 0.1 N hydrochloric acid. Then these solutions werewell mixed to effect reaction and allowed to stand at 37 C. for 18hours. And then, 5 volumes of 99% ethyl alcohol was added to the mixtureand allowed to stand for 30 minutes.

The resulting white precipitate was collected by centrifugation at 5,000r.p.m. for 10 minutes, then added with twice the volume of 99% ethylalcohol under agitation and again subjected to centrifugation of 5,000r.p.m. for 10 minutes. The precipitate thus collected is recrystallizedfrom ethyl ether to obtain 10.1 g. of the desired product as a colorlesscrystalline powder.

EXAMPLE 3 0.5 g. of cadaverine (C H N molecular weight 102.18) and 5 g.of deoxyribonucleic acid having molecular weight of ca. 300,000extracted from thymus gland of calf were separately dissolved into 0.01M sodium chloride solutions, and the solutions thus obtained were mixedto react and then allowed to stand at 37 C. for 36 hours. The reactionmixture was treated similarly as in Example 1 to obtain 5.4 g. of thedesired product as a colorless crystalline powder.

EXAMPLE 4 l g. of polylysine, i.e. a basic polyamino acid, and g. ofdeoxyribronucleic acid having molecular weight of ca. 200,000 extractedfrom thymus of calf were separately dissolved in 0.01 mol sodiumchloride solutions, and the solutions thus obtained were mixed to reactand allowed to stand at 37 C. for 18 hours. The reaction mixture wastreated similarly as in Example 1 to obtain 10.1 g. of the desiredproduct as a colorless crystalline powder.

EXAMPLE 5 0.1 g. of polyarginine, i.e. a basic polyamino acid, and 5 g.of deoxyribonucleic acid having molecular weight of ca. 500,000extracted from salmon sperm were separately dissolved in 0.01 M sodiumchloride solution, and the solutions thus obtained were mixed to reactand then allowed to stand at 37 C. for 18 hours. The reaction mixturewas treated similarly as in Example 1 to obtain 5.0 g. of the desiredproduct as a colorless crystalline powder.

What is claimed is:

1. A compound comprising a complex formed between deoxyribonucleic acidand a basic polyamine selected from the group consisting of spermine,spermidine and cadaverine or a basic polyamino acid selected from thegroup consisting of polylysine and polyarginine.

2. A compound as claimed in claim 1 in which said deoxyribonucleic acidhas a molecular weight within the range of 200,000 to 500,000.

3. A compound as claimed in claim 2 in which the weight ratio of acid tobasic polyamine or basic polyamino acid in said complex is within therange of 10:1 to :1.

4. A process for the preparation of a compound as claimed in claim 1consisting essentially of the steps of dissolving deoxyribonucleic acidin a first dilute sodium chloride solution or a dilute sodium chloridesolution containing sodium citrate; dissolving a basic polyamineselected from the group consisting of spermine, spermidine andcadaverine or a basic polyamino acid selected from the group consistingof polylysine and polyarginine in a second dilute sodium chloridesolution or a dilute sodium chloride solution containing sodium citrate;adjusting the pH value of each of the resulting solutions to 7.0; mixingthe pH-adjusted solutions to effect reaction between thedeoxyribonucleic acid and the basic polyamine or basic polyamino acid;allowing the reaction mixture to stand at 37 C. for 6 to 36 hours;adding ethanol to the pH- adjusted reaction mixtures and collecting theprecipitate thus formed by centrifugation.

References Cited UNITED STATES PATENTS 2,710,860 6/1955 Ruskin 260211.5R 3,089,869 5/ 1963 Mauvernay 26021l.5 R 3,155,647 11/1964 Dutcher etal. 260-2115 R 3,215,687 11/1965 Tsuchiya et al. 260211.5 R 3,287,35111/1966 Cantineau et al. 260211.5 R 3,300,476 1/1967 Zahn et al.260-2115 R 3,314,937 4/1967 Vendrely et al. 260--211.5 R

LEWIS GOTIS, Primary Examiner I. R. BROWN, Assistant Examiner US. Cl.X.R. 26021l.5 R; 424-

